Fetal tissue has been transplanted into human patients to treat Parkinsonism and other neurodegenerative diseases and is a promising treatment for many other conditions, including neurotrauma and injuries to the spinal cord. Fetal cells are very useful because they are multipotent, meaning that they have the potential to turn into many different kinds of specialized cells, for example a neuron (a brain cell) or a liver cell. A multipotent cell is sometimes called a stem cell. A cell that changes from a multipotent state into a specialized cell is said to differentiate into a differentiated (i.e., specialized) cell. Once a cell differentiates into a specialized cell it does not naturally return to a multipotent state. Thus, any cell that is not yet fully committed to a particular specialized cell type is referred to hereinafter as a “stem cell.”
Transplantation of fetal tissue has had limited success. One technical limitation is that implanted fetal cells do not necessarily differentiate into the desired cell type. For example, a fetal cell put into the brain of a Parkinson's patient does not necessarily become a type of neuron that benefits the patient, such as a dopaminergic neuron. In addition, significant moral, ethical, and technological issues make a non-fetal source of cells desirable.
Scientists have learned that stem cells exist in mammals at all stages of development, including the adult stage. Adult stem cells are more specialized than fetal stem cells but have the natural potential to become one of a wide variety of cell types. The stem cell types are commonly named according to the tissue where they reside: for example, bone marrow stem cells, epidermal (skin) stem cells, or central nervous system stem cells. Many hospitals routinely capture bone marrow stem cells from patients undergoing chemotherapy. The cells are preserved outside of the body during treatment and subsequently implanted following treatment.
A substantial body of literature describes therapies based on introducing cells into patients. These therapies include treatments of Alzheimer's disease and Parkinson's disease. Such therapies are described, for example, in “Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain” by F. H. Gage et al., Proceedings of the National Academy of Science U.S.A. 92:11879-83 (1995); “Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain” by R. A. Fricker et al., Journal of Neuroscience, 19:5990-6005 (1999); and “Self-repair in the brain” by A. Bjorklund and O. Lindvall, Nature 405:892-3,895 (2000).
Parkinson's Disease and Intracerebral Transplantation
Parkinson's disease is a neurodegenerative disease characterized by profound loss of dopaminergic neurons in the substantia nigra. The loss of dopaminergic neurons in the substantia nigra results in the degeneration of the nigrostriatal dopamine system that regulates motor function. This, in turn, leads to motor dysfunction, consisting of poverty and slowness of voluntary movements, tremor, stooped posture, rigidity, and gait disturbance. There has been no cure for Parkinson's disease.
Modern knowledge of the pathogenesis of Parkinson's disease indicates that successful functional restoration can be achieved by replacing the lost dopamine in the damaged area of the brain. This understanding inspired attempts to replace dopamine by grafting dopamine producing cells, for example, fetal tissue rich in dopaminergic neurons, cells from the adrenal medulla, or dopaminergic neurons from another species, such as pigs, to the degenerated striatum. The ability of intracerebral grafts to induce behavioral recovery in brain-damaged recipients rests on the multitude of trophic, neurohumoral, and synaptic mechanisms that may allow the implanted tissue to promote host brain function and repair. To what extent intracerebral implants can be functionally integrated with the host brain, particularly in man, is still poorly understood and remains a topic for further clinical investigation. The chances for extensive integration may be greatest for very small neural grafts or cell suspensions. Re-innervation of these small, solid grafts seems to be a function of the rapid availability of a rich vascular bed or contact with cerebral spinal fluid. Evidence suggests that the grafts may be revascularized very quickly, perhaps within hours, especially as cell suspensions (Leigh et al., 1994). The results of these types of cell replacement therapies are encouraging, but heterogeneity of transplanted cells, risks for immunological rejection and other problems related to the transplantable material have raised numerous concerns about cell-based therapies.
Fetal Dopaminergic Neurons
Clinical data clearly demonstrate that fetal mesencephalic dopamine neurons obtained from a human fetus can survive and function in the brains of patients with Parkinson's disease. Unfortunately, functional recovery after transplantation has been only partial, and both the reproducibility and efficacy of the procedure must be significantly improved. Nevertheless, early publications on transplantation of fetal dopaminergic neurons have demonstrated success. In a study from Great Britain an initial 12 patients had fetal tissue from a single donor placed stereotactically into the caudate (Hitchcock et al., 1989). The patients showed improvement within one week and levodopa dosage (the more traditional therapy) was reduced by 29% within the first three months and by 24% within the first six months. Follow-up on nine patients demonstrated a 29% improvement in the Webster rating scale at three months and a 42% improvement at six months. Other early reports indicated similar improvement (Freed et al., 1989). Since the late 1980's, when human fetal tissue transplants began, it has been estimated that over 500 patients with Parkinson's disease have received fetal implants. The results in two series of experiments in the United States have been reported. Freed et al. have made observations in seven patients followed from 12 to 46 months after mesencephalic fetal transplants (Freed et al., 1992). Two of these patients had unilateral implants into the caudate and putamen and five had bilateral implants into the putamen only. Long-term moderate improvement was reported, and the Sinemet dosage was substantially reduced. The improvement was related to the presence or absence of immunosuppressant drugs. In another series of experiments the improvement appeared to be more mild (Spencer et al., 1992). These less impressive results may be related to the cryopreservation of the transplanted fetal tissue and the older age of the tissue. A major conclusion from these results is that implantation of fetal dopamine-rich mesencephalic tissue can lead to a therapeutically valuable, sustained improvement in motor function in patients with idiopathic Parkinson's disease (see Lang and Lozano, 1998).
The main limitations of current fetal cell-transplantation procedures are the practical, ethical and safety issues related to the use of fetal tissue. The large number of fetal dopaminergic neurons that are needed to obtain therapeutic effects in patients restricts the applications of transplantation procedures to highly specialized medical centers. Current transplantation techniques result in survival of 5-20% of the transplanted neurons. Consequently, cells from 3 to 5 fetuses yield only 100,000-150,000 surviving dopaminergic neurons (Lindvall, 1997). Animal experiments have demonstrated that inhibition of cell death by caspase inhibitors, free radical scavengers, and neurotrophic factors may increase dopamine neuron survival 2 to 3 fold (Sinclair et al., 1996, Zawada et al., 1998, Schierle et al., 1999). Application of these additions to human clinical protocols may increase the cell survival and reduce the number of fetal cells required for efficient therapeutic effect. The main focus of current research is developing techniques to improve survival and growth of transplanted dopaminergic neurons.
Autologous Adrenal Medulla Grafts
Backlund and his group in Stockholm, Sweden started human transplants based on experimentation by collaborators in Lund, Sweden. In their experiments, cell suspensions were stereotactically placed into the caudate. Although their results were not spectacular, probably because they implanted relatively pure suspensions of neurons without associated glial cells, their experiments opened up the field to other investigators. Subsequently, Dr. Ignacio Madrazo and Dr. Drucker-Colin described a series of 54 patients with Parkinson's disease who showed marked improvement in their disease some months after they had received a transplant of autologous adrenal medulla to the caudate nucleus of their brain (Madrazo et al., 1987). Their success seems stem from changing their protocol so that they implant very small pieces of adrenal gland (and, more recently, fetal grafts that have open access to cerebral spinal fluid so that graft viability is maintained until neovascularization). Following Madrazo's results, Allen et al., at Vanderbilt University and Jiao et al. in Beijing, China, reported on multiple patients with severe Parkinson's disease who had improved after undergoing a technique very similar to Madrazo's.
CNS Stem Cell Propagation, Differentiation and Transplantation
An alternative approach to treating a neurodegenerative disease such as Parkinson's disease is to take tissue from a patient or donor, isolate the central nervous stem cells from the tissue, cause the stem cells to differentiate into the desired type of neurons, and implant the neurons into the appropriate region in the patient's brain. This approach is referred to as autologous transplantation because the cells are taken from a patient and implanted into the same patient. The same process could be applicable to many neuronal diseases and disorders in addition to Parkinson's disease. For example, the process could be used to treat spinal cord damage.
Following removal of an appropriate tissue sample, the process would involve three steps: isolation, propagation, and differentiation. In the isolation step, stem cells are preferably separated from all the other cells in a tissue sample. Alternatively, the tissue may be placed in a chemical environment that preferentially facilitates the growth of stem cells. In the propagation step the stem cells are kept alive and preferably encouraged to multiply, for example from a few cells into tens of thousands of cells. In the differentiation step, the cells are preferably caused to develop into the type of cell that is suitable for the application. For example, in the case of a Parkinson's patient, at least a portion of the stem cells are preferably caused to develop into neurons. Following differentiation, the differentiated cells may be implanted into the patient. In the case of a Parkinson's patient, the differentiated cells would preferably be implanted in the patient's brain. For the treatment of spinal cord injury, the differentiated cells would be placed in the spinal cord at or near the site of injury.
Although such treatments have been contemplated, there continues to be a need for actual compositions and methods for isolation, propagation, and differentiation of stem cells for treating nervous system pathologies.